Embryo culture, freezing and thawing protocols
Fertilization and embryo culture were carried out in Quinn’s IVF
sequential medium suite
(Quinn’s,
SAGE, USA) with adding 10% human serum substitute (Quinn’s, SAGE, USA)
in 5% O2, 5% CO2, 90%
N2 and saturated humidity. Follicles were aspirated
transvaginally under ultrasound guidance at 36 h post hCG injected.
Cumulus-oocyte complexes and sperm were mixed at 39-40 h post hCG
injected. For ICSI, the cumulus cells were removed enzymatically and the
sperm was injected at about 42 h after hCG injected. The fertilization
results were evaluated 16–18 h after fertilization and embryos were
cultured individually in 25 μl microdroplet of cleavage medium covered
with oil. Cleavage-stage embryos were scored and graded every day for
blastomere number, cleavage plane and degree of fragmentation. Available
embryos were vitrified using
vitrification media (Kitazato
Biopharma, Shizuoka, Japan) on day 3 after egg collection (D3). Briefly,
embryos were transferred from the culture medium to the center of the
equilibration solution surface and remained in the media for about 7
minutes at room temperature. Then, the embryos were moved into
vitrification solution and blown and inhaled at different positions.
After 40-50 s in this solution, the embryos were placed on the Cryotop
(Kitazato Biopharma, Shizuoka, Japan) and rapidly placed into liquid
nitrogen. Embryos were thawed using vitrification media (Kitazato
Biopharma, Shizuoka, Japan). Briefly, Cryotop was taken rapidly from
liquid nitrogen and immersed in a 300 μl droplet of thawing solution
(TS) at 37℃. At the end of 1 minute, the embryos were transferred to a
300 μl droplet of diluent solution (DS) at room temperature for 3
minutes. Then, the embryos were
shifted to 300 μl droplets of washing solution 1 (WS1) and washing
solution 2 (WS2) at room temperature for 5 minutes, respectively.
Finally, the embryos were transferred to a 30 μl droplet of blastocyst
medium and cultured for 2-3 h or 18-20 h for transfer.